Probing Local Protein Environments in Adenylate Kinase with 4‐Cyano‐L‐Phenylalanine

Abstract

Unnatural amino acids (UAAs) containing vibrational reporters, such as nitrile, azide, and nitro groups, can be utilized to effectively probe local protein environments. Specifically, the nitrile symmetric stretching frequency of 4‐cyano‐L‐phenylalanine (pCNF) is sensitive to local protein hydration and occurs in a relatively clear region of the infrared spectrum. In addition, the magnitude of change in this vibrational frequency over a given temperature range is dependent on the local protein environment of the nitrile group of pCNF. Finally, the UAA pCNF can also be site‐specifically incorporated into proteins with high efficiency and fidelity using the Amber codon suppression methodology. Here, pCNF was incorporated into several different local protein environments in the enzyme Adenylate Kinase (AK). The successful incorporation was verified by time‐of‐flight mass spectrometry. These results in addition to the linear temperature‐dependent IR analysis of these constructs to extract the hydration state of the nitrile group of pCNF in each of these AK constructs will be presented.